Recent advances in the therapeutic efficacy of hepatocyte growth factor gene‐modified mesenchymal stem cells in multiple disease settings

Abstract Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co‐transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune‐related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs' clinical application based on our understanding of the HGF gene and MSC therapy.

Hepatocyte growth factor (HGF) is a pleiotropic factor primarily secreted by mesenchymal cells 15 that was first identified and cloned in the 1980s. 16,17 HGF has mitogenic, motogenic, anti-apoptotic, morphogenic and immune regulation activities, 18,19 which can prevent fibrosis, apoptosis and inflammation, and promote angiogenesis in multiple conditions. 20 It has been proven that MSCs' therapeutic efficacy completely or partially depends on the secretion of HGF. [21][22][23][24][25] In 2003, our group first reported research about HGF gene-modified MSCs, in which the MSCs' therapeutic efficacy on myocardial ischaemia was improved by HGF gene modification. 26 Since then, dozens of studies about HGF gene-modified MSCs have been conducted. This review summarizes the characteristics of HGF gene-modified MSCs (HGF/MSCs). In addition, the mechanisms of their enhanced therapeutic efficacy were analysed, thus giving some insights into their clinical application.

| CHAR AC TERIS TI C S OF HG F/MSC IN VITRO
Up to now, seven kinds of HGF gene modification vectors have been used in preclinical studies, namely Ad-HGF, adeno-associated virus vector carrying HGF gene (AAV-HGF), lentivirus vector carrying HGF gene (Lenti-HGF), retrovirus vector carrying HGF (Retro-HGF), HGF plasmid, transcription activator-like effector nucleases (TALEN) system and gene-delivery nano-system (Table S1) MSCs. 27 For HGF/MSC application, it is important to clarify whether the HGF gene modification has changed these characteristics.

| THER APEUTI C EFFI C AC Y OF HG F/MSC S IN PRECLINIC AL S TUD IE S
HGF/MSCs showed a synergic therapeutic effect of MSC and HGF (Table 2). Both MSC therapy and HGF protein/gene therapy were beneficial to angiogenesis, organ structure recovery, organ function recovery and anti-fibrosis. In contrast, HGF/MSC therapy was more effective than either alone. 26,44,47,50,[57][58][59] Table S1. The main mechanisms of HGF/MSC therapy are summarized in Figure 1, and they are further demonstrated below.

| PROMOTING ENG R AF TMENT AND TISSUE REPAIRMENT
The proliferation and migration activities of the transplanted MSCs are related to their in vivo engraftment efficiency. HGF is a mitogen factor involved in organ development and regeneration. 20 In preclinical studies, HGF gene modification enhanced the therapeutic effect of MSCs on the organ structure recovery through enhancing soft tissue re-epithelialization, restoring cell-cell connection and promoting hard tissue regeneration. Epithelial cells are widely distributed between the external and internal surfaces of the host, thus playing an important role in organ physiological homeostasis. 61 HGF/MSCs promoted the activities and reduced the apoptosis of epithelial cells in the diseased organs, including the injured intestine, 54 transplanted trachea, 55 involute thymi 37 and burned skin. 62 Cells are connected by tight junctions and gap junctions, while the tight junctions are only found among epithelial cells. 63 Zonula occludens-1 (ZO-1) is a major component of tight junctions, and connexin 43 (Cx43) is a kind of gap junction protein. 64 The expression of ZO-1 54 and Cx43 65 was upregulated by HGF/MSCs more efficiently than only MSCs. Other than enhancing re-epithelialization and restoring the cell-cell connection, the bone regeneration was promoted by HGF/MSCs more than only MSCs in the diseased microenvironments. 38,40,48

| PROMOTING ANG IOG ENE S IS
All tissues should be nurtured by the extensive networks formed by blood vessels. The progression of various diseases correlates with tissue destruction, such as necrosis and ischaemic and inflammatory diseases. 66 It was reported that HGF/MSCs promoted vascular endothelial cell proliferation and blood vessel regeneration more efficiently than MSCs and HGF protein. 46,48,51,58,59,67,68 The endothelial

| PROMOTING ANTI -FIB ROS IS EFFEC T
Fibrosis is defined by the accumulation of excess ECM components, which can affect any organ and is responsible for up to 45% of all deaths in the industrialized world. 70 It has been reported that HGF/ MSCs could reduce fibrosis efficiently in various diseased organs, including the heart, 68,71 lung, 29,55,72-74 liver, 47,52,56,[75][76][77][78] kidney, 45,49 bladder, 79  and Smad3, is negatively regulated by Smad7 expression. 80 In the rats with liver fibrosis, the HGF/UC-MSC transplant downregulated the expression of TGF-β1, Smad2 and Smad3 more efficiently than UC-MSCs, 78 suggesting that the advantageous therapeutic efficacy of HGF/MSCs might depend on the TGFβ/Smad signalling pathway.

Both MSCs and HGF possess anti-inflammatory properties.
Inflammation is a complex set of interactions in response to traumatic, infectious, post-ischaemic, toxic or autoimmune injuries, which can lead to persistent tissue damage by leukocytes, lymphocytes or collagen. 81 It was reported that HGF/MSCs were beneficial to the survival of the graft in the host. 43

| PROMOTING ANTI -AP OP TOS IS EFFEC T
HGF gene modification not only could enhance the anti-apoptosis potential of MSCs in the microenvironments of hypoxia or inflammation in vitro, but it also could enhance their suppression effect on the apoptosis of parenchymal cells in vivo, such as cardiomyocytes, 34,69,82 lung epithelial cells, 28,29 hepatocytes, 44,47,75 renal cells 30 and intestinal epithelial cells. 54 It was reported that the expression of caspase-3 was suppressed by HGF/MSCs more than when only MSCs were used. 30

| PROMOTING ANTI -OXIDATION EFFEC T
Oxidative stress is implicated in various chronic/degenerative diseases, resulting in macromolecular damage. 87 There are two kinds of oxidant compounds, namely reactive oxygen species (ROS) and reactive nitrogen species (RNS), which introduce various oxidative insults to lipids, proteins and nucleic acids, with consequences ranging from subtle modulation of cell signal transduction processes to apparent biomolecular damage and cell death. 88 The antioxidant system is composed of nonenzymatic antioxidants and enzymatic antioxidants. The nonenzymatic antioxidants are low molecular weight compounds, including glutathione (GSH), vitamin C and β-carotene.
The enzymatic antioxidants can be divided into two groups: the antioxidant response element-driven enzymes and primarily or constitutively acting antioxidant enzymes, such as superoxide dismutase (SOD), catalase and GSH peroxidase. 89 It was reported that HGF/ MSCs could upregulate the expression of SOD and downregulate the expression of malondialdehyde (MDA), GSH and γ-glutamyl cysteine synthetase (γ-GCS) more efficiently than the MSCs when transplanted in vivo. 28,31 MDA is an indicator for lipid peroxidation, and γ-GCS is a rate-limiting enzyme of GSH synthesis within the cell. Therefore, HGF/MSCs could reduce oxidative stress by decreasing lipid peroxidation, probably through their ability to promote the activity of SOD and the synthesis of GSH.

| THER APEUTI C EFFI C AC Y OF HG F/MSC APPLI C ATI ON IN A CLINI C AL S TU DY
Silicosis is an irreversible disease characterized by lung fibrosis. A clinical study on HGF/MSCs therapy for silicosis concluded that the administration of HGF/BM-MSCs was safe and effective in some patients with silicosis. Briefly, HGF/BM-MSCs were prepared by transfecting autologous BM-MSCs with plasmid HGF. Then, HGF/ BM-MSCs were administered intravenously to four patients with pulmonary silicosis at a dose of 2 × 10 6 cells/kg weekly for three consecutive weeks. Two patients had dexamethasone-relievable fever after the administration, but no other abnormal symptoms were observed after the treatment for 6 months. The lung function indicators, such as forced vital capacity (FVC), the forced expiratory volume averages at the first second (FEV1) and the arterial blood oxyhemoglobin saturation (SpO 2 ), were improved; the ratios of peripheral blood CD4 + /CD8 + cell concentrations were increased; the serum IgG levels were decreased to the normal range; and the average ceruloplasmin level was slightly decreased, indicating an improvement of lung function and a reduction of inflammation.
Furthermore, the absorption of the nodular lesion was observed after treatment for 12 months in 2 patients, suggesting structural healing from the silicotic fibrosis. 90

| D ISCUSS I ON AND PER S PEC TIVE S
Gene-modified stem cells could be applied to the next generation of cell-based therapies. How to screen for the gene and cell source, the modification process, and the indications for the modified cell suitable for the therapies are three basic questions that need to be answered in gene-modified stem cell therapy. In Table 2 Col I in the trabeculae, but increased its expression in medullary cavities 48 ; decreased the expression of α-SMA in fibrosis (Table 2), but increased its expression in the cavernous tissue. 35 The reason might be that the behaviour and activities of the transplanted HGF/MSCs could be affected by the surrounding physical (e.g. stiffness, elasticity, viscosity, hypoxia, fluid shear stress, hydrostatic pressure, bioelectricity and microgravity), chemical (e.g.

cells, nonparenchymal cells and immune cells) microenvironments.
To determine the changes in cell behaviours and activities be- writing -review and editing (lead).

FU N D I N G I N FO R M ATI O N
The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.